A Single-Center 18-Year Series of 73 Cases of Metaplastic Carcinoma of the Breast.

Aim: To examine the clinical management of metaplastic breast cancer (MeBC), particularly the role of chemotherapy. Methods: This retrospective study included patients with MeBC (n = 73) from a tertiary breast cancer center: the "Centre des Maladies du Sein of the CHU de Québec-Université Laval." The specimens were reviewed by two pathologists. Patient and tumor characteristics, systemic therapy (neoadjuvant and adjuvant), disease-free survival (DFS), and overall survival (OS) were recorded. Results: The median follow-up was 57.2 months. The mean tumor size was 39.5 ± 32.1 (range, 1-200) mm. Most were in grade 3 (75.3%), without evidence of clinical nodal involvement (75.3%), and triple-negative (79.5%). Chemotherapy was given to 49 (67.1%) patients. Thirty-seven patients (50.7%) underwent a mastectomy, and 22/37 (59.5%) received radiotherapy. Adjuvant chemotherapy was given to 36 patients (49.3%), and nine (12.3%) patients were treated with neoadjuvant chemotherapy. The 5-year OS and DFS rates were 60.2% and 66.8%. Among the nine patients who received neoadjuvant chemotherapy, three (33.3%) achieved a partial response, three (33.3%) had stable disease, and three (33.3%) had disease progression. The use of chemotherapy, especially in the adjuvant setting, had a significant positive effect on 5-year OS (P=0.003) and 5-year DFS (P=0.004). Nodal involvement was associated with worse OS (P=0.049) but similar DFS (P=0.157). Lumpectomy was associated with better 5-year OS (P < 0.0001) and DFS (P=0.0002) compared with mastectomy. Conclusion: MeBC represents a rare heterogeneous group of malignancies with poor prognosis. Adjuvant chemotherapy was associated with improved OS and DFS. Patients should be carefully selected for neoadjuvant chemotherapy.

HER2 Immunohistochemistry in Invasive Micropapillary Breast Carcinoma: Complete Assessment of an Incomplete Pattern.

CONTEXT.­: Invasive micropapillary carcinoma (IMPC) is a rare variant of breast carcinoma, composed of avascular morula-like tumor clusters surrounded by stromal spaces, which can affect the HER2 immunohistochemical (IHC) staining pattern. The 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 testing guideline suggests moderate to intense but incomplete (basolateral) staining be considered equivocal. OBJECTIVES.­: To perform a detailed assessment of HER2 IHC staining patterns in IMPC. DESIGN.­: Hematoxylin-eosin and HER2 IHC slides were retrospectively reviewed to assess the morphology and HER2 IHC characteristics of IMPC. The 2018 ASCO/CAP guideline was applied. RESULTS.­: The cohort consisted of 187 IMPCs from 181 patients with median age of 58 years. Homogeneous (≥90%) micropapillary component was found in 40% (75 of 187) of cases. Receptor profile was as follows: 75% (140 of 187) ER+ HER2-, 19% (37 of 187) ER+ HER2+, 4% (7 of 187) ER- HER2+, and 2% (3 of 187) ER- HER2-. Of 26 cases with HER2 IHC 3+, 65% (17 of 26) showed a basolateral staining pattern with strong intensity. HER2 fluorescence in situ hybridization (FISH) showed amplification in 26% (17 of 66) of HER2 IHC equivocal cases: 76% (13 of 17) showed basolateral staining pattern and 24% (4 of 17) complete staining, with weak to moderate (2), moderate (14), or moderate to strong (1) intensity. CONCLUSIONS.­: The most frequent staining pattern was basolateral, seen in 49% of cases, including 65% HER2 IHC positive and 76% HER2 IHC equivocal/FISH amplified. If a basolateral pattern and weak to moderate staining is observed in IMPC, alternative testing should be performed to confirm the HER2 status.

Solid Pseudo-Papillary Tumor Mimicking as Complicated Pseudocyst: Multimodality Imaging and Pathological Correlation.

Pancreatic neoplasm is very rare in the pediatric population. Malignant tumors represent less than 0.2% of pediatric cancer-related mortality. Pancreas lesions can be from exocrine or endocrine origin or present themselves as cystic masses. Clinical, biological, and radiological findings usually are sufficient to establish diagnosis, but in some cases, they may be misleading. We present the case of a young patient presenting a pancreatic mass where anatomical and metabolic characteristics of the lesion were discordant to the final diagnosis.

Epithelial Myoepithelial Carcinoma of the Nasal Cavity: Clinical, Histopathological, and Immunohistochemical Distinction of a Case Report.

BACKGROUND: Epithelial myoepithelial carcinomas (EMCs) are rare low-grade salivary gland tumors. Here, we report the case of a 75-year-old man presenting with an oncocytic variant of EMC of the nasal cavity, initially diagnosed as an oncocytoma. METHODS: Our patient underwent functional sinus surgery in 2012. On pathology, an oncocytic neoplasm was found in the right nasal cavity, characterized by fragments of uniform bland oncocytic cells with bilayered arrangement of nuclei. Immunohistochemical stains demonstrated biphasic cells: luminal epithelial and basal cell-type myoepithelial cells. The tumor was best diagnosed as an oncocytoma. In 2015, the patient presented with a recurrent right inferior turbinate lesion, compatible with oncocytic EMC. RESULTS: The patient underwent oncological surgery and received adjuvant radiotherapy. He had no disease recurrence. CONCLUSION: Different variants of EMCs exist, such as oncocytic EMC. EMCs should be treated aggressively because they can be locally invasive, recur, and give rise to distant metastases.

Regulation of human Dicer by the resident ER membrane protein CLIMP-63.

The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of microRNAs, which are known to regulate messenger RNA (mRNA) translation. In order to improve our understanding of the molecular context in which Dicer functions and how it is regulated in human cells, we sought to expand its protein interaction network by employing a yeast two-hybrid screening strategy. This approach led to the identification and characterization of cytoskeleton-linking endoplasmic reticulum (ER) membrane protein of 63 kDa (CLIMP-63) as a novel Dicer-interacting protein. CLIMP-63 interacts with Dicer to form a high molecular weight complex, which is electrostatic in nature, is not mediated by RNA and is catalytically active in pre-microRNA processing. CLIMP-63 is required for stabilizing Dicer protein and for optimal regulation of a reporter gene coupled to the 3' untranslated region of HMGA2 mRNA in human cells. Interacting with a portion of the luminal domain of CLIMP-63 and within minutes of its synthesis, our results suggest that Dicer transits through the ER, is glycosylated and can be secreted by cultured human cells with CLIMP-63. Our findings define CLIMP-63 as a novel protein interactor and regulator of Dicer function, involved in maintaining Dicer protein levels in human cells.

Detection of human Dicer and Argonaute 2 catalytic activity.

The microRNA (miRNA)-guided RNA silencing pathway is a central and well-defined cellular process involved in messenger RNA (mRNA) translational control. This complex regulatory process is achieved by a well orchestrated machinery composed of a relatively few protein components, among which the ribonuclease III (RNase III) Dicer and Argonaute 2 (Ago2) play a central role. These two proteins are essential and it is of particular interest to measure and detect their catalytic activity under various situations and/or conditions. In this chapter, we describe different protocols that aim to study and determine the catalytic activity of Dicer and Ago2 in cell extracts, immune complexes, and size-fractionated cell extracts. Another protocol aimed at assessing miRNA binding to Ago2 is also described. These experimental approaches are likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases.

Existence of a microRNA pathway in anucleate platelets.

Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion, which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes, which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system.

Protein components of the microRNA pathway and human diseases.

MicroRNAs (miRNAs) are key regulators of messenger RNA (mRNA) translation known to be involved in a wide variety of cellular processes. In fact, their individual importance is reflected in the diseases that may arise upon the loss, mutation or dysfunction of specific miRNAs. It has been appreciated only recently that diseases may also develop when the protein components of the miRNA machinery itself are affected. The core enzymes of the major protein complexes involved in miRNA biogenesis and function, such as the ribonucleases III (RNases III) Drosha and Dicer as well as Argonaute 2 (Ago2), appear to be essential. However, the accessory proteins of the miRNA pathway, such as the DiGeorge syndrome critical region gene 8 (DGCR8) protein, Exportin-5 (Exp-5), TAR RNA binding protein (TRBP) and fragile X mental retardation protein (FMRP), are each related, in various ways, to specific genetic diseases.

Protein interactions and complexes in human microRNA biogenesis and function.

Encoded in the genome of most eukaryotes, microRNAs (miRNAs) have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. The aim of this review is to present this process as the integration of a succession of specialized molecular machines exerting well defined functions. The nuclear microprocessor complex initially recognizes and processes its primary miRNA substrate into a miRNA precursor (pre-miRNA). This structure is then exported to the cytoplasm by the Exportin-5 complex where it is presented to the pre-miRNA processing complex. Following pre-miRNA conversion into a miRNA:miRNA* duplex, this complex is assembled into a miRNA-containing ribonucleoprotein (miRNP) complex, after which the miRNA strand is selected. The degree of complementarity of the miRNA for its messenger RNA (mRNA) target guides the recruitment of the miRNP complex. Initially repressing its translation, the miRNP-silenced mRNA is directed to the P-bodies, where the mRNA is either released from its inhibition upon a cellular signal and/or actively degraded. The potency and specificity of miRNA biogenesis and function rely on the distinct protein x protein, protein x RNA and RNA:RNA interactions found in different complexes, each of which fulfill a specific function in a well orchestrated process.

The Saccharomyces cerevisiae Arf3 protein is involved in actin cable and cortical patch formation.

We show that Arf3p, a member of the ADP ribosylation family, is involved in the organization of actin cables and cortical patches in Saccharomyces cerevisiae. Profilin-deficient cells (pfy1Delta) have severe growth defects and lack actin cables. Overexpression of ARF3 restores actin cables and corrects growth defects in these cells. Cells deficient for the cortical patch proteins Las17p and Vrp1p have growth defects and a random cortical patch distribution. Overexpression of ARF3 in las17Delta and in vrp1Delta cells partially corrects growth defects and restores the polarized distribution of cortical patches. The N-terminal glycine, a myristoylation site in Arf3p, is necessary for its suppressor activity. arf3Delta cells show a random budding pattern. Overexpression of BNI1, GEA2 or SYP1, three genes involved in actin cytoskeleton formation, restores the normal axial budding pattern of arf3Delta cells. BUD6 is a polarity gene and GEA2 is involved in retrograde transport and the organization of the actin cytoskeleton. We have identified genetic interactions between ARF3 and BUD6, and between ARF3 and GEA2. Both double mutant strains have actin cytoskeleton defects. Our results support a role for ARF3 in cell polarity and the organization of the actin cytoskeleton.

Regulatory RNAs: future perspectives in diagnosis, prognosis, and individualized therapy.

With potentially up to 1000 microRNAs (miRNAs) present in the human genome, altogether regulating the expression of thousands of genes, one can anticipate that miRNAs will play a significant role in health and disease. Deregulated protein expression induced by a dysfunctional miRNA-based regulatory system is thus expected to lead to the development of serious, if not lethal, genetic diseases. A relationship among miRNAs, Dicer, and cancer has recently been suggested. Further investigations will help establish specific causal links between dysfunctional miRNAs and diseases. miRNAs of foreign origin, e.g., viruses, may also be used as specific markers of viral infections. In these cases, miRNA expression profiles could represent a powerful diagnostic tool. Regulatory RNAs may also have therapeutic applications, by which disease-causing genes or viral miRNAs could be neutralized, or functional miRNAs be restored. Will bedside miRNA expression profiling eventually assist physicians in providing patients with accurate diagnosis, personalized therapy, and treatment outcome?

MicroRNAs in gene regulation: when the smallest governs it all.

Encoded by the genome of most eukaryotes examined so far, microRNAs (miRNAs) are small approximately 21-nucleotide (nt) noncoding RNAs (ncRNAs) derived from a biosynthetic cascade involving sequential processing steps executed by the ribonucleases (RNases) III Drosha and Dicer. Following their recent identification, miRNAs have rapidly taken the center stage as key regulators of gene expression. In this review, we will summarize our current knowledge of the miRNA biosynthetic pathway and its protein components, as well as the processes it regulates via miRNAs, which are known to exert a variety of biological functions in eukaryotes. Although the relative importance of miRNAs remains to be fully appreciated, deregulated protein expression resulting from either dysfunctional miRNA biogenesis or abnormal miRNA-based gene regulation may represent a key etiologic factor in several, as yet unidentified, diseases. Hence is our need to better understand the complexity of the basic mechanisms underlying miRNA biogenesis and function.

A role for GEA1 and GEA2 in the organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Profilin is an actin monomer-binding protein implicated in the polymerization of actin filaments. In the budding yeast Saccharomyces cerevisiae, the pfy1-111 rho2delta double mutant has severe growth and actin cytoskeletal defects. The GEA1 and GEA2 genes, which code for paralog guanosine exchange factors for Arf proteins, were identified as multicopy suppressors of the mutant phenotype. These two genes restored the polarized distribution of actin cortical patches and produced visible actin cables in both the pfy1-111 rho2delta and pfy1delta cells. Thus, overexpression of GEA1 or GEA2 bypassed the requirement for profilin in actin cable formation. In addition, gea1 gea2 double mutants showed defects in budding and in actin cytoskeleton organization, while overexpression of GEA1 or GEA2 led to the formation of supernumerary actin cable-like structures in a Bni1p/Bnr1p-dependent manner. The ADP-ribosylation factor Arf3p may be a target of Gea1p/Gea2p, since overexpression of ARF3 partially suppressed the profilin-deficient phenotype and a deletion of ARF3 exacerbated the phenotype of a pfy1-111 mutant. Gea1p, Gea2p, Arf1p, and Arf2p but not Arf3p are known to function in vesicular transport between the endoplasmic reticulum and the Golgi. In this work, we demonstrate a role for Gea1p, Gea2p, and Arf3p in the organization of the actin cytoskeleton.